PPARGC1A/NRF1/TFAM通路核心基因在湖羊睾丸发育过程中的表达变化
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摘要
[目的]本试验旨在探究PPARGC1A/NRF1/TFAM通路核心基因在湖羊性成熟前后的表达变化。[方法]选取体况良好和系谱清楚的雄性湖羊18只(3和9月龄,各9只),颈静脉采血后进行阉割处理。ELISA检测血样中睾酮(T)、瘦素、胰岛素及胰岛素样生长因子Ⅰ(IGF-Ⅰ)的浓度;免疫组化法检测睾丸组织中PPARGC1A/NRF1/TFAM通路相关蛋白的表达定位; RT-qPCR和Western blot检测睾丸组织中PPARGC1A/NRF1/TFAM通路相关基因与蛋白的表达变化。[结果]与3月龄湖羊相比,9月龄湖羊外周血中T、瘦素、胰岛素及IGF-Ⅰ的浓度显著升高(P<0.05)。免疫组化法检测发现PPARGC1A、NRF1和TFAM蛋白在3月龄和9月龄湖羊睾丸组织的多种细胞内均呈时空特异性表达。此外,与3月龄湖羊相比,9月龄湖羊睾丸组织中PPARGC1A、NRF1和TFAM m RNA和蛋白表达水平均显著升高(P<0.05)。[结论]PPARGC1A/RNF1/TFAM通路相关基因可能与湖羊睾丸发育和性成熟过程密切相关。
[Objectives]The objective of this study was to elucidate the localization and expression of the key PPARGC1 A/NRF1/TFAM pathway genes in the testis of prepubertal( 3-month-old) and postpubertal( 9-month-old) Hu sheep. [Methods]Eighteen male Hu sheeps with good body conditions and clear pedigree were selected for this experiment( 3-and 9-month-old,n = 9). The testis was collected after blood collection. The concentrations of testosterone( T),leptin,insulin and insulin-like growth factorⅠ( IGF-Ⅰ) in the blood samples was detected by ELISA; cellular localization of PPARGC1 A/NEF1/TFAM pathway key proteins in testis was detected by immunohistochemistry; and the expression patterns of the PPARGC1 A/NRF1/TFAM pathway core genes in testis tissue were detected by RT-qPCR and Western blot. [Results]Compared with the 3-month-old Hu sheep,the concentrations of the T,leptin,insulin,and IGF-Ⅰ in the peripheral blood of 9-month-old Hu sheep significantly increased( P < 0.05,respectively). Immunohistochemistry results showed an age-related and tissue-specific expressions of PPARGC1 A/RNF1/TFAM pathway key proteins in the testis of 3-month-old and 9-month-old Hu sheep. In addition,the m RNA of PPARGC1 A,NRF1 and TFAM and its protein expressions in the testis of 9-month-old Hu sheep were significantly higher than those in 3-month-old Hu sheep( P< 0.05). [Conclusions]The PPARGC1 A/RNF1/TFAM pathway may be closely associated with testicular development and sexual maturation of Hu sheep.
[Objectives]The objective of this study was to elucidate the localization and expression of the key PPARGC1 A/NRF1/TFAM pathway genes in the testis of prepubertal( 3-month-old) and postpubertal( 9-month-old) Hu sheep. [Methods]Eighteen male Hu sheeps with good body conditions and clear pedigree were selected for this experiment( 3-and 9-month-old,n = 9). The testis was collected after blood collection. The concentrations of testosterone( T),leptin,insulin and insulin-like growth factorⅠ( IGF-Ⅰ) in the blood samples was detected by ELISA; cellular localization of PPARGC1 A/NEF1/TFAM pathway key proteins in testis was detected by immunohistochemistry; and the expression patterns of the PPARGC1 A/NRF1/TFAM pathway core genes in testis tissue were detected by RT-qPCR and Western blot. [Results]Compared with the 3-month-old Hu sheep,the concentrations of the T,leptin,insulin,and IGF-Ⅰ in the peripheral blood of 9-month-old Hu sheep significantly increased( P < 0.05,respectively). Immunohistochemistry results showed an age-related and tissue-specific expressions of PPARGC1 A/RNF1/TFAM pathway key proteins in the testis of 3-month-old and 9-month-old Hu sheep. In addition,the m RNA of PPARGC1 A,NRF1 and TFAM and its protein expressions in the testis of 9-month-old Hu sheep were significantly higher than those in 3-month-old Hu sheep( P< 0.05). [Conclusions]The PPARGC1 A/RNF1/TFAM pathway may be closely associated with testicular development and sexual maturation of Hu sheep.
引文
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[11]崔玉倩.睾酮对胰岛β-细胞胰岛素分泌和凋亡的影响及机制[D].济南:山东大学,2012.Cui Y Q.Effects and mechanisms of testosterone on insulin secretion and cell apoptosis inβcells[D].Jinan:Shandong University,2012(in Chinese with English abstract).
[12]林晖榕,陈慎仁,傅玉才,等.不同月龄大鼠胰岛细胞衰老及胰岛素表达的研究[J].中国病理生理杂志,2009,25(1):180-182.Lin H R,Chen S R,Fu Y C,et al.Research on senile cellular senescence and insulin expression in rats of different months[J].Chinese Journal of Pathophysiology,2009,25(1):180-182(in Chinese with English abstract).
[13]Priyadarshini L,Yadav A K,Singh H S,et al.Role of leptin in physiology of animal reproduction:a review[J].Agricultural Reviews,2015,36(3):235-240.
[14]Gamba M,Pralong F P.Control of GnRH neuronal activity by metabolic factors:the role of leptin and insulin[J].Mol Cell Endocrinol,2006,254/255:133-139.
[15]李松,李建波,李金凤.1岁以内不同胎龄儿生长速度的比较及与FGF21、瘦素的相关性[J].中国妇幼保健,2017,32(2):308-310.Li S,Li J B,Li J F.Comparison of growth velocity of infants of different gestational age under one year and the correlation with FGF21 and leptin[J].Maternal and Child Health Care of China,2017,32(2):308-310(in Chinese with English abstract).
[16]Spicer L J.Leptin:a possible metabolic signal affecting reproduction[J].Domestic Animal Endocrinology,2015,21(4):251-270.
[17]Guerriero G,Trocchia S,Abdel-Gawad F K,et al.Roles of reactive oxygen species in the spermatogenesis regulation[J].Front Endocrinol(Lausanne),2014,5:56-61.
[18]Anderson C M,Mendelson C R.Regulation of steroidogenesis in rat Leydig cells in culture:effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin[J].Arch Biochem Biophys,1985,238(2):378-387.
[19]Allen J A,Shankara T,Janus P,et al.Energized,polarized,and actively respiring mitochondria are required for acute Leydig cell steroidogenesis[J].Endocrinology,2006,147(8):3924-3935.
[20]Mulder H.Transcribingβ-cell mitochondria in health and disease[J].Molecular Metabolism,2017,6(9):1040-1051.
[21]Rato L,Duarte A I,Tomas G D,et al.Pre-diabetes alters testicular PGC1-α/SIRT3 axis modulating mitochondrial bioenergetics and oxidative stress[J].Biochimica Et Biophysica Acta,2014,1837(3):335-344.
[22]Yu Y X,Han Y L,Niu R Y,et al.Ameliorative effect of VE,IGF-Ⅰ,and h CG on the fluoride-induced testosterone release suppression in mice Leydig cells[J].Biological Trace Element Research,2018,181(1):95-103.
[23]Martinezabundis E,Rajapurohitam V,Gertler A,et al.Identification of functional leptin receptors expressed in ventricular mitochondria[J].Mol Cell Biochem,2015,408(1/2):155-162.
[24]Pinto-Fochi M E,Pytlowanciv E Z,Reame V,et al.A high-fat diet fed during different periods of life impairs steroidogenesis of rat Leydig cells[J].Reproduction,2016,152(6):795-808.
[25]Pedram A,Razandi M,Wallace D C,et al.Functional estrogen receptors in the mitochondria of breast cancer cells[J].Molecular Biology of the Cell,2006,17(5):2125-2137.
[2]Ohkura S,Uenoyama Y,Yamada S,et al.Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone(Gn RH)secretion in female rats[J].Peptides,2009,30(1):49-56.
[3]Mayer C,Adam M,Walenta L,et al.Insights into the role of androgen receptor in human testicular peritubular cells[J].Andrology,2018,6(5):756-765.
[4]Chang H,Guillou F,Taketo M M,et al.Overactive beta-catenin signaling causes testicular sertoli cell tumor development in the mouse[J].Biology of Reproduction,2009,81(5):842-849.
[5]Oldknow K J,Seebacher J,Goswami T,et al.Follistatin-like 3(FSTL3)mediated silencing of transforming growth factorβ(TGFβ)signaling is essential for testicular aging and regulating testis size[J].Endocrinology,2013,154(3):1310-1320.
[6]郑九嘉,楼哲丰,郑蔚虹,等.线粒体呼吸功能与精子活力、核DNA损伤的相关性分析[J].中国细胞生物学学报,2012,34(1):34-40.Zheng J J,Lou Z F,Zheng W H,et al.Analysis of mitochondria respiratory function associated with motility of sperm and nucleus DNA damage[J].Chinese Journal of Cell Biology,2012,34(1):34-40(in Chinese with English abstract).
[7]Lu Z,Xu X,Hu X,et al.PGC-1 alpha regulates expression of myocardial mitochondrial antioxidants and myocardial oxidative stress after chronic systolic overload[J].Antioxid Redox Signal,2010,13(7):1011-1022.
[8]岳根华,谢成侠,程瑞禾,等.湖羊及考力代公羔生殖系统发育与精子发生的研究[J].南京农业大学学报,1993,16(3):69-73.DOI:10.7685/j.issn.1000-2030.1993.03.014.Yue G H,Xie C X,Cheng R H,et al.Study on the development of reproductive system and spermatogenesis in Hu sheep and Corriedale male lambs[J].Journal of Nanjing Agricultural University,1993,16(3):69-73(in Chinese with English abstract).
[9]Kim J H,Park S J,Kim T S,et al.Testosterone production by a Leydig tumor cell line is suppressed by hyperthermia-induced endoplasmic reticulum stress in mice[J].Life Sciences,2015,146:184-191.
[10]Nobue K,Wanzhu J,Gen W,et al.Age-related changes of reproductive hormones in young Meishan boars[J].Journal of Reproduction&Development,2006,52(5):651.
[11]崔玉倩.睾酮对胰岛β-细胞胰岛素分泌和凋亡的影响及机制[D].济南:山东大学,2012.Cui Y Q.Effects and mechanisms of testosterone on insulin secretion and cell apoptosis inβcells[D].Jinan:Shandong University,2012(in Chinese with English abstract).
[12]林晖榕,陈慎仁,傅玉才,等.不同月龄大鼠胰岛细胞衰老及胰岛素表达的研究[J].中国病理生理杂志,2009,25(1):180-182.Lin H R,Chen S R,Fu Y C,et al.Research on senile cellular senescence and insulin expression in rats of different months[J].Chinese Journal of Pathophysiology,2009,25(1):180-182(in Chinese with English abstract).
[13]Priyadarshini L,Yadav A K,Singh H S,et al.Role of leptin in physiology of animal reproduction:a review[J].Agricultural Reviews,2015,36(3):235-240.
[14]Gamba M,Pralong F P.Control of GnRH neuronal activity by metabolic factors:the role of leptin and insulin[J].Mol Cell Endocrinol,2006,254/255:133-139.
[15]李松,李建波,李金凤.1岁以内不同胎龄儿生长速度的比较及与FGF21、瘦素的相关性[J].中国妇幼保健,2017,32(2):308-310.Li S,Li J B,Li J F.Comparison of growth velocity of infants of different gestational age under one year and the correlation with FGF21 and leptin[J].Maternal and Child Health Care of China,2017,32(2):308-310(in Chinese with English abstract).
[16]Spicer L J.Leptin:a possible metabolic signal affecting reproduction[J].Domestic Animal Endocrinology,2015,21(4):251-270.
[17]Guerriero G,Trocchia S,Abdel-Gawad F K,et al.Roles of reactive oxygen species in the spermatogenesis regulation[J].Front Endocrinol(Lausanne),2014,5:56-61.
[18]Anderson C M,Mendelson C R.Regulation of steroidogenesis in rat Leydig cells in culture:effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin[J].Arch Biochem Biophys,1985,238(2):378-387.
[19]Allen J A,Shankara T,Janus P,et al.Energized,polarized,and actively respiring mitochondria are required for acute Leydig cell steroidogenesis[J].Endocrinology,2006,147(8):3924-3935.
[20]Mulder H.Transcribingβ-cell mitochondria in health and disease[J].Molecular Metabolism,2017,6(9):1040-1051.
[21]Rato L,Duarte A I,Tomas G D,et al.Pre-diabetes alters testicular PGC1-α/SIRT3 axis modulating mitochondrial bioenergetics and oxidative stress[J].Biochimica Et Biophysica Acta,2014,1837(3):335-344.
[22]Yu Y X,Han Y L,Niu R Y,et al.Ameliorative effect of VE,IGF-Ⅰ,and h CG on the fluoride-induced testosterone release suppression in mice Leydig cells[J].Biological Trace Element Research,2018,181(1):95-103.
[23]Martinezabundis E,Rajapurohitam V,Gertler A,et al.Identification of functional leptin receptors expressed in ventricular mitochondria[J].Mol Cell Biochem,2015,408(1/2):155-162.
[24]Pinto-Fochi M E,Pytlowanciv E Z,Reame V,et al.A high-fat diet fed during different periods of life impairs steroidogenesis of rat Leydig cells[J].Reproduction,2016,152(6):795-808.
[25]Pedram A,Razandi M,Wallace D C,et al.Functional estrogen receptors in the mitochondria of breast cancer cells[J].Molecular Biology of the Cell,2006,17(5):2125-2137.